us pharma presentation on clone screen strategy for monoclonality using solentim cell metric cld
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Efficient Screening Strategies to Address Monoclonality & Reduce TimelinesChaomei He, Associate Director , Momenta Pharmaceuticals, Inc. USAIBC 4th Annual Cell Line Development & Engineering Asia
Outline
Copyright © 2015. Momenta Pharmaceuticals, Inc. All rights reserved
Introducing Momenta Pharmaceuticals
Challenges in selection and clonal isolation: addressing
monoclonality
Review of latest technologies
Clonal cell lines selection using Cell Image System
Combining image processing and document tracking system for
effective clone-picking
Summary
Overview of Momenta Pharmaceuticals
Founded in 2001; IPO 2004
Located in Cambridge, Massachusetts, USA
~260 employees; >75% in scientific development
Expertise in high-resolution analytics, biological
characterization, and process engineering
Current business areas:
Complex drug generics
Biosimilars and potentially interchangeable biologics
Novel medicines
675 West Kendall StCambridge, MA
320 Bent StCambridge, MA
Copyright © 2015. Momenta Pharmaceuticals, Inc. All rights reserved
PROGRAMSPRECLINICAL/PROCESS
DEVELOPMENTCLINICAL1
ANDA/BLA/NDAFILED
MARKETED
ComplexGenerics1
Enoxaparin SodiumInjection (Generic Lovenox®)*
GlatopaTM (Generic 20 mg/mL Copaxone®)*
M356 (Generic 40 mg/mL Copaxone®)*
Biosimilars
M923 Adalimumab(HUMIRA®)**
M834 Abatacept (ORENCIA®)
M511 Bevacizumab (AVASTIN®)
6 Early Stage BiosimilarPrograms
NovelDrugs
Necuparanib - pancreaticcancer
hsIVIg (Hyper-sialylated IVIg)
SIF3 (Selective Immuno-modulator of Fc Receptors)
Anti-FcRn Antibody
Approved
ANDA Accepted
CTA Accepted/PK Study
Phase 2
1Clinical safety/efficacy trials have not been required for these complex generic drug applications
Momenta Product Portfolio: Broad Application of Thorough Characterization Across Complex Generics, Biosimilars, and Novel Drugs
*In collaboration with Sandoz**In collaboration with Baxter
Copyright © 2015. Momenta Pharmaceuticals, Inc. All rights reserved
- Vector Design
- Transfection
- Stable Pools
- 2-6 weeks
- Clones selection & screening- Scale up- Batch / Fed-Batch screening - 8-12 weeks
- Evaluation in bioreactors
- 4-6 weeks
- Cell Line Stability Study (Preliminary)
- 10-16 weeks
-Lead clone selection
-Generation of Research Cell Bank
A Typical Cell Line Development Flow Diagram for a Protein
Copyright © 2015. Momenta Pharmaceuticals, Inc. All rights reserved
Cell line development is a complex multistep process leading to the generation of a Research Cell Bank which is the precursor of Master Cell Bank: The properties of the selected clone will significantly impact the
process and the product itself Changing the cell line in the midst of a development program is
expensive and time consuming
Selection and Clonal Isolation
Copyright © 2015. Momenta Pharmaceuticals, Inc. All rights reserved
Proteins based therapeutic drugs have achieved great success in clinic, sales grew exponentially in the last decade, and this trend is expected to continue
Generation of clonal cell lines leading up to Research Cell Bank is typically the rate limiting step towards an IND
“The cell substrate is the transfected cells containing the desired sequences, which has been cloned from a single cell progenitor”ICH Guideline Q5D
Challenges in Streamlining Selection and Clonal Isolation
Copyright © 2015. Momenta Pharmaceuticals, Inc. All rights reserved
Efficient processes are essential to reduce timelines and costs Optimize each step from transfection to final clone by
defining criteria of success, implementing high throughput technologies and avoiding repeats. Build Quality in each step.
Documentation to support regulatory filings required from Day 1 From detailed information on the host cell line to
demonstration of monoclonality
Demonstration of monoclonality requires proper design, execution and documentation in the generation of the Research Cell Bank
Regulatory Requirements For Demonstration of Clonality
Anne B. Tolstrup – Boehringer Ingelheim Cell Line Development and Engineering, Berkeley 2014
How to Achieve Monoclonality ?
Two rounds of Limited Dilution Cloning (LDC) add significant time and expenses to the cell line development efforts
WCBP 2014 Sarah Kennett, Ph.D., FDA
Copyright © 2015. Momenta Pharmaceuticals, Inc. All rights reserved
How to Achieve Monoclonality ?
Use of a cell imaging system can reduce timelines and costs:
System needs to provide appropriate resolution and deliver documentation of the images
Operator needs to review images following wells from day zero Human intervention is required to ensure monoclonality
Significant IT requirements (huge files, need for high speed analysis, etc.)
WCBP 2014 Sarah Kennett, Ph.D., FDA
Copyright © 2015. Momenta Pharmaceuticals, Inc. All rights reserved
Latest Technologies For Single Cell Cloning
FACS AriaTM (BD Biosciences) Cloning by FACS at 1 cell/well
• Not all well has single cell, some well contain more than one cell
• Additional Cell image documentation is required to prove clonality
ClonePix FL System (Molecular devices), picking colonies in semi-solid medium
• Additional round of ClonePix is required to prove clonality
CloneSelectTM Imager (Molecular devices)
Cell MetricsTMCLD System (Solentim)
Octet High Throughput titer measurement (96, 384-well)
Copyright © 2015. Momenta Pharmaceuticals, Inc. All rights reserved
The Power of Imaging : Example of Single Colony Generated From a Single Cell Progenitor
Day 7
Day 3
Day 2
Day 1
Day 0
Copyright © 2015. Momenta Pharmaceuticals, Inc. All rights reserved
The Power of Imaging: Example of Single Colony Starting From Two Cells
Day 0 Day 1 Day 7
Copyright © 2015. Momenta Pharmaceuticals, Inc. All rights reserved
The Power of Imaging: Example of a False Negative
No cell was detected on day zero, but cells appeared in later days
No evidence that cells were derived from a single cell Resolution is critical
Day 0 Day 1
No cell
Copyright © 2015. Momenta Pharmaceuticals, Inc. All rights reserved
Percentage of Wells Contain Single Cell
Program Wells with Single Cell
Total # of Colonies
% Single Cell/ Total # of Colonies
Protein - A 783 1693 46%
Protein - B 721 2066 35%
Protein - C 436 1108 40%
Based on Poisson distribution, the probability of a single cell/well at seeding density of 0.5 cell/well dilution is 77%• In reality, the actual percentage of single cell/well is much lower
With a cell imaging system, day zero image can be captured• Select cell lines that demonstrate derivation from a single cell
progenitor The use of a cell imaging system allows for one round of limiting
dilution cloning, when performed using an appropriate dilution and acquiring images from day zero
Copyright © 2015. Momenta Pharmaceuticals, Inc. All rights reserved
Documentation of Monoclonality
Cell imaging system records original well ID of the single cell progenitor, and images associated with this well from day zero
An electronic automation tracking system can increase throughput and improve traceability• Well ID of the single cell progenitor - scale up - bioreactor – stability study
– lead clone selection – RCB – MCB/WCB
16 Copyright © 2015. Momenta Pharmaceuticals, Inc. All rights reserved
Example of Electronic Tracking
Record
Copyright © 2015. Momenta Pharmaceuticals, Inc. All rights reserved
Summary
Generation of a manufacturing cell line is typically the rate limiting step for filing an IND
The requirements for generating and testing manufacturing cell lines are well described in the literature
Demonstration of monoclonality is an important part of the overall Regulatory package
Monoclonality can be demonstrated by: 2 rounds of Limited Dilution Cloning
1 round of Limited Dilution Cloning with a cell imaging system
Cell Imaging system must have the appropriate resolution to allow for documentation of the initial doubling steps: from 1 cell to 2 cells to 4 cells.. Images need to be reviewed by operators
High demand on IT systems
Automation of well tracking increases throughput and improves traceability
Elimination of a Limited Dilution Cloning can reduce timelines significantly while meeting Regulatory requirements regarding monoclonality
Copyright © 2015. Momenta Pharmaceuticals, Inc. All rights reserved
Acknowledgments
LDC and Documentation
Billy Stuart Marcio Voloch
Megha Kapur
Copyright © 2015. Momenta Pharmaceuticals, Inc. All rights reserved
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